Schematic representation of processing of endogenous Ihh and expression of Ihh in gene-modified human stromal cells. (A) Expression of SH2, SH3, and GFP in control and Ihh stromal cells. Control or Ihh-transduced stromal cells (Ihh stromal cells) were sorted after gene transduction. Postsorting purities of the GFP⁺ control and Ihh stromal cells were 96.7% and 93.3%, respectively. Cells were gated on the basis of forward and side light scatter to exclude debris. (B) Expression of Ihh mRNA analyzed by RT-PCR specific for vector-derived Ihh and endogenous Ihh: lane 1, control stromal cells; lane 2, Ihh stromal cells; lane 3, H₂O. (C) Immunoblot analysis of Ihh in supernatant (1) and cytosol (2) of human stromal cells. The LTC medium of the stromal cells was replaced in the serum-free X-VIVO10 medium after washing with PBS, and supernatants were collected for 72 hours after the culture. Ihh was immunolabeled with the 5E1 antihedgehog antibody. Lane 1, control stromal cells; lane 2, Ihh stromal cells.